Angiogenesis is a precisely regulated process which coordinates the assembly and differentiation of numerous cell types to form the arteries, capillaries and veins of the pre-existing vascular bed. The primitive vasculature is composed of an endothelial plexus, which require the recruitment of pericytes and vascular smooth muscle cells by soluble growth factors secreted by endothelial cells to pattern the vessels into arteries and veins (Risau, “Mechanisms of Angiogenesis,” Nature 386:671-674 (1997)). In the final steps of vessel formation, the newly formed endothelial cells are stabilized by the extracellular matrix, the formation of a basement membrane and ensheathment with pericytes and smooth muscle cells. Numerous polypeptide growth factors have been implicated in initiating vasculogenesis and angiogenic sprouting, including fibroblast growth factors (bFGF and FGF-2), vascular endothelial growth factor (VEGF), and the angiopoietins (Darland et al., “Blood Vessel Maturation: Vascular Development Comes of Age,” J. Clin. Invest. 103:167-168 (1999); Ferrara et al., “The Biology of Vascular Endothelial Growth Factor,” Endocrin. Rev. 18:4-25 (1997)). In addition, platelet derived growth factor B (PDGF-BB), angiopoietin-1 (ang-1), ephrin B2, and TGFβ have been shown to regulate later aspects of the angiogenesis process, in the recruitment of mural cells, and in the patterning of the vascular bed (Yancopoulos et al., “Vasculogenesis, Angiogenesis and Growth Factors: Ephrins Enter the Fray at the Border,” Cell 93:661-664 (1998); Lindahl et al., “Pericyte Loss and Microaneurysm Formation in the PDGF-B-deficient mice,” Science 277:242-245 (1997); Dickman et al., “Defective Haematopoiesis and Vasculogenesis in Transforming Growth Factor Beta 1 Knock Out Mice,” Development 121:1845-1854 (1995); Yang et al., “Angiogenesis Defects and Mesenchymal Apoptosis in Mice Lacking SMAD5,” Development 126:1571-1580 (1999)). Very little is known about growth factors which regulate the stabilization and survival of the mature vasculature, although angiopoietin-1 has been proposed as a candidate molecule. Of these factors, only VEGF has been rigorously tested for its ability to initiate angiogenesis in adults in preclinical and clinical trials (Ferrara et al., “The Biology of Vascular Endothelial Growth Factor,” Endocrin. Rev. 18:4-25 (1997); Mack et al., “Biologic Bypass With the Use of Adenovirus-Mediated Gene Transfer of the Complementary Deoxyribonucleic Acid for Vascular Endothelial Growth Factor 121 Improves Myocardial Perfusion and Function in the Ischemic Porcine Heart,” J. Thoracic and Cardiovascular Surgery, 115:168-176 (1998); Losordo et al., “Gene Therapy for Myocardial Angiogenesis: Initial Clinical Results with Direct Myocardial Injection of phVEGF165 as Sole Therapy for Myocardial Ischemia,” Circulation 98:2800-2804 (1998)). Although delivery of VEGF by gene transfer can induce an angiogenic response in ischemic tissues, exogenous VEGF induces the formation of fragile, dilated and malformed vessels (Springer et al., “VEGF Gene Delivery to Muscle: Potential Role for Vasculogenesis in Adults,” Molecular Cell 2:549-558 (1998); Drake et al., “Exogenous Vascular Endothelial Growth Factor Induces Malformed and Hyperfused Vessels During Embryonic Development,” Proc. Natl. Acad. Sci. 92:7657-7661 (1995)). In addition, recent studies suggest that the endothelial cells of postnatal vessels may become independent of VEGF for their continued survival within several weeks of birth in rodents (Gerber et al., “VEGF is Required for Growth and Survival in Neonatal Mice,” Development 126:1149-1159 (1999)). Thus, the ultimate endpoint is the definition of the cellular steps and molecular sequences that direct and maintain microvascular assembly leading to therapeutic targets for repair and adaptive remodeling.
In recent studies, the roles of the neurotrophins in regulating cardiovascular development and modulating the vascular response to injury have been investigated (Donovan et al., “Neurotrophin-3 is Required for Mammalian Cardiac Development: Identification of an Essential Nonneuronal Neurotrophin Function,” Nature Genetics 14:210-213 (1996); Donovan et al., “Neurotrophin and Neurotrophin Receptors in Vascular Smooth Muscle Cells: Regulation of Expression in Response to Injury,” A. J. Path. 147:309-324 (1995); Kraemer et al., “NGF Activates Similar Intracellular Signaling Pathways in Vascular Smooth Muscle Cells as PDGF-BB But Elicits Different Biological Responses,” Arteriol. Thromb. And Vasc. Biol. 19:1041-1050 (1999)). The neurotrophins today consist of a family of five related polypeptide growth factors: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophins 3, 4 (also referred to as neurotrophin 5), and 6 (NT-3, NT-4, NT-6) (Lewin et al., “Physiology of the Neurotrophins,” Ann. Rev. Neuro. 19:289-317 (1996)). These structurally related proteins mediate their actions on responsive neurons by binding to two classes of cell surface receptor (Lewin et al., “Physiology of the Neurotrophins,” Ann. Rev. Neuro. 19:289-317 (1996)). The low affinity neurotrophin receptor, p75, binds all neurotrophins and modulates signaling initiated by the second class of neurotrophin receptors, the trk family of receptor tyrosine kinases (what was originally identified as the trk tyrosine kinase receptor is now referred to as trk A, one member of the trk family of receptors). Trk A, trk B, and trk C tyrosine kinases serve as the receptors for NGF, BDNF, and NT-3, respectively, and trk B can also be activated by NT-4.
NT-3 initiates a number of trophic effects on neurons expressing its receptor, trk C, ranging from mitogenesis, promotion of survival, or differentiation, depending on the developmental stage of the target cells (Chalazonitis, “Neurotrophin-3 as an Essential Signal for the Developing Nervous System,” Molecular Neurobiology 12:29-53 (1996)). The reported sites of action of NT-3 reside primarily in the peripheral nervous system (PNS), various areas of the central nervous system (CNS), and in the enteric nervous system (ENS). Id. Analyses of the phenotypes of transgenic mice lacking NT-3 or injection of embryos with a blocking antibody have revealed the essential role of NT-3 in development of specific populations of the PNS, and in particular of proprioceptive, nodose, and auditory sensory neurons and of sympathetic neurons. Id. The actions of NT-3 also extend to modulation of transmitter release at several types of synapses in the periphery as well as in the adult CNS. Id.
NT-4 acts via the trk B receptor and supports survival of primary somatic and visceral sensory neurons (Erickson et al., “Mice Lacking Brain-Derived Neurotrophic Factor Exhibit Visceral Sensory Neuron Losses Distinct from Mice Lacking NT4 and Display a Severe Developmental Deficit in Control of Breathing,” J. Neurosci. 16:5361-5371 (1996)). The major visceral sensory population, the nodose-petrosal ganglion complex (NPG), requires BDNF and NT-4 for survival of a full complement of neurons, however, only one functional NT-4 allele is required to support survival of all NT-4-dependent neurons. Id. NT-4 appears to have the unique requirement of binding to p75 for efficient signaling and retrograde transport in neurons (Ibáñez, “Neurotrophin-4: The Odd One out in the Neurotrophin Family,” Neurochemical Research 21:787-793 (1996)). In addition, while all other neurotrophin knock-outs have proven lethal during early postnatal development, mice deficient in NT-4 have so far only shown minor cellular deficits and develop normally to adulthood.
Trk B receptors and BDNF are highly expressed by central and peripheral neurons, and gene ablation studies have demonstrated the critical role of trk B and BDNF in neuronal differentiation and survival, with gene targeted animals exhibiting abnormalities in cerebellar function and respiratory drive (Lewin et al., “Physiology of the neurotrophins,” Ann. Rev. Neuro. 19:289-317 (1996); Jones et al., “Targeted Disruption of the BDNF Gene Perturbs Brain and Sensory Neuron Development But Not Motor Neuron Development,” Cell 76:989-999 (1994); Erickson et al., “Mice Lacking Brain-Derived Neurotrophic Factor Exhibit Visceral Sensory Neuron Losses Distinct From Mice Lacking NT4 and Display a Severe Developmental Deficit in Control of Breathing,” J. Neurosci. 16:5361-5371 (1996); Schwartz et al., “Abnormal Cerebellar Development and Foliation in the BDNF (−/−) Mice Reveals a Role for Neurotrophins in CNS Patterning,” Neuron 19:269-281 (1997)).
However, the BDNF:trk B receptor system is expressed at high levels in nonneuronal tissues, including muscle, lung, kidney, heart and the vasculature, where its biological functions are unclear (Donovan et al., “Neurotrophin and Neurotrophin Receptors in Vascular Smooth Muscle Cells: Regulation of Expression in Response to Injury,” A. J. Path. 147:309-324 (1995); Timmusk et al., “Widespread and Developmentally Regulated Expression of Neurotrophin-4 mRNA in Rat Brain and Peripheral Tissues,” Eur. J. Neurosci. 5:605-613 (1993); Hiltunen et al., “Expression of mRNAs for Neurotrophins and Their Receptors in Developing Rat Heart,” Circ. Res. 79:930-939 (1996); Scarisbrick et al., “Coexpression of the mRNAs for NGF, BDNF and NT-3 in the Cardiovascular System of Pre- and Post-Natal Rat,” J. Neurosci. 13:875-893 (1993)). Prior studies have identified roles for the related neurotrophin, NT-3, and its receptor, trk C, in regulating cardiac septation and valvulogenesis (Donovan et al., “Neurotrophin-3 is Required for Mammalian Cardiac Development: Identification of an Essential Nonneuronal Neurotrophin Function,” Nature Genetics 14:210-213 (1996); Tessarollo et al., “Targeted Deletion of all Isoforms of the trk C Gene Suggests the Use of Alternate Receptor by its Ligand Neurotrophin-3 in Neural Development and Implicates trk C in Normal Cardiogenesis,” Proc. Natl. Acad. Sci. USA 94:14766-014781 (1997). In addition, it has been demonstrated that BDNF and trk B are expressed by vascular smooth muscle cells of the adult aorta, and expression of this ligand:receptor system is upregulated in neointimal cells following vascular injury (Donovan et al., “Neurotrophin-3 is Required for Mammalian Cardiac Development: Identification of an Essential Nonneuronal neurotrophin Function,” Nature Genetics 14:210-213 (1996)). However, the biological actions of BDNF and related neurotrophins in cardiovascular function and development have not been assessed.
The present invention is directed to functions of the neurotrophins and the trk receptor family related to vascular biology.